PROJECT SUMMARY/ABSTRACT The actin cytoskeleton is crucial for cellular properties and behaviors including shape, migration, and division, as well as for cell-cell connections and fusion during animal development. Because of these numerous essential functions in cell physiology, actin dysfunction is also a common contributor to pathogenesis, for example in inflammation, cardiovascular disease, cancer metastasis, and microbial infection. Despite many years of study, however, understanding how actin assembly is regulated and harnessed in the cytoplasm and nucleus for intracellular events, cellular behaviors, and cell-cell interactions remains a key outstanding problem in cell biology. My lab has taken a distinctive approach to address this important gap in knowledge, which is to examine the interactions between infectious microbes and the actin cytoskeleton as a window into actin regulation and function. Our approach leverages the fact that many infectious microbes colonize host cells through their ability to target actin, and builds on numerous examples of how studying the interactions between microbes and host cells has enhanced our understanding of cytoskeleton dynamics, as well as membrane trafficking, cell cycle regulation, protein recycling, and cell death. The research described in this MIRA application makes use of microbes as tools to address three fundamental cell biological questions: (1) How is actin polymerization at membranes regulated and mobilized to drive movement? (2) How and why is actin transported into and polymerized within the nucleus for gene expression, nuclear organization, intranuclear movement, and nuclear envelope dynamics? (3) How is actin polymerization in plasma membrane protrusions harnessed to induce cell-cell fusion? We will investigate these questions using three model microbes, each of which mobilizes actin in a manner that makes it a unique and powerful instrument: Mycobacterium marinum as a tool to understand the regulation of actin assembly at membranes to drive intracellular movement; the baculovirus Autographa californica multiple nucleopolyhedrovirus as a tool to understand the regulation and function of actin in the nucleus; and Burkholderia thailandensis as a tool to understand the role of actin in cell-cell fusion. By leveraging our expertise in both microbiology and cell biology, deploying a synergistic combination of microbial and mammalian genetic methods, and employing biochemical, biophysical and imaging approaches, we are uniquely positioned to advance the field forward. Our results will enhance our understanding of the mechanisms of actin regulation and may provide new insights into diagnosing, treating, and preventing diseases associated with actin dysfunction including inflammatory and cardiovascular diseases, cancer, and microbial infection.